Research this year was focused on: (1) the molecular basis of the localization of Acanthamoeba myosin IC (AMIC) to specific membranes, (2) the role of S-adenosylhomocysteine hydrolase (SAHH) in chemotaxis, and (3) the properties of Tyr53-phosphorylated actin (pY53-actin).[unreadable] [unreadable] (1) The 466-residue tail of AMIC has an N-terminal basic region followed by a Gly/Pro/Ala-rich (GPA) region, an SH3 domain, and a C-terminal GPA region. We had shown previously (Hwang et al., 2007) that the basic region contains a plekstrin homology (PH) domain, and speculated that the PH domain might be involved in the high-affinity binding of AMIC to membranes and acidic phospholipid vesicles, which is known to be mediated through the basic region (Doberstein & Pollard, 1992). [unreadable] [unreadable] We found that AMIC binds with higher affinity to unilamellar phosphatidylcholine vesicles containing phosphoinositol bisphosphate (PIP2)than to vesicles containing phosphatidylserine (PS). The tighter binding is fully attributable to the 5-fold higher negative charge of PIP2, for example, AMIC binds with equal affinity to vesicles containing 5% PIP2 or 25% PS. As expected, the basic region in the tail is both necessary and sufficient for binding. Ongoing work with appropriate deletion mutants will determine if the PH domain in the basic region is required for high affinity binding, or if binding is due simply to the basic charge. [unreadable] [unreadable] We found also that AMIC binds with 10-fold higher affinity to vesicles containing 5% PIP2+25% PS than to vesicles containing only 25% PS, which suggests that AMIC might be targeted to PIP2-enriched regions of membranes in the amoebae. Consistent with this hypothesis, we find that AMIC and PIP2 co-localize at endocytic cups, pseudopods, membrane ruffles and the leading edge of motile cells. [unreadable] [unreadable] (2) Last year we reported that S-adenosylhomocysteine hydrolase (SAHH), an essential component of the trans-methylation pathway in all eukaryotic cells, is diffuse in the cytoplasm of resting cells but localizes with polymerized actin at the front of both chemotaxing Dictyostelium amoebae and human neutrophils (Shu et al., 2006). These results add SAHH to the list of proteins that relocate to either the front or rear of a chemotaxing cell, and suggest a role for methylation in chemotaxis, which was further supported by the observation that tubercidin (an adenosine analogue that inhibits SAHH) inhibits chemotaxis.[unreadable] [unreadable] More extensive work this year showed that tubercidin has little, if any, effect on cAMP-induced chemotaxis of individual Dictyostelium amoebae, but delays the formation of cell streams, an essential step in the developmental cycle of Dictyostelium. Attempts to isolate SAHH-knockout cells were unsuccessful, but Dictyostelium cell lines were created that express only 25% of the normal amount of SAHH. The phenotype of the SAHH-knockdown cells is different than the phenotype of wild-type cells exposed to tubercidin, suggesting that tubercidin inhibition of chemotaxis may not be due to inhibition of SAHH. This is not surprising given the many biochemical pathways that an adenosine analogue like tubercidin might inhibit.[unreadable] [unreadable] (3) Tyrosine53-phosphorylated actin (pY53-actin) forms late in the Dictyostelium developmental cycle, and also when cells in the amoeboid stage are subjected to stress. Last year, we purified pY53-actin and showed that Tyr53-phosphorylation increases actins critical concentration, reduces the rate of polymerization, substantially inhibits nucleation and elongation from the pointed-end of actin filaments, and moderately reduces the rate of elongation from the barbed-end (Liu et al., 2006). Under polymerization conditions, pY53-actin forms small oligomers that are converted to typical long filaments upon addition of myosin subfragment 1, which is activated normally by filamentous pY53-actin.[unreadable] [unreadable] Efforts this year were concentrated on obtaining sufficient pY53-actin for our collaborators at the University of Pennsylvania, Drs. Roberto Dominguez and Kyuwon Baek, to determine its atomic structure by X-ray crystallography. The crystal structure of unphosphorylated actin in a ternary complex with profilin I and a VASP peptide has been analyzed. Small crystals of pY53-actin have been obtained, but crystals of pY53-actin suitable for diffraction studies have not yet been obtained.